Rosemary (rosmarinus officinalis l.) denominated ki937

ABSTRACT

The present invention relates generally to a rosemary plant and, more specifically, to a proprietary clonal plant line KI937 that hyper-accumulates carnosic acid.

RELATED APPLICATION

This application claims priority to the benefit of U.S. PatentApplication Ser. No. 62/840,754, filed Apr. 30, 2019.

FIELD OF THE INVENTION

The present invention relates generally to a rosemary plant and, morespecifically, to a proprietary clonal plant line denominated KI937 thatproduces desirable antioxidants such as carnosic acid.

BACKGROUND OF THE INVENTION

Rosemary (Rosmarinus officinalis L.) is a member of the Lamiaceae familyof plants, which encompasses over 500 species that have been widely usedin traditional medicine. The antioxidant properties of members of theLamiaceae family (including rosemary) have been identified within thepast 4-5 decades and have demonstrated superior antioxidant activity ina variety of applications (Cuppett et al., 1997). Rosmarinus officinaliscontains monoterpenes, sesquiterpene chemical composition, diterpenes,triterpenoids, flavonoids, fatty acids, amino acids and branchedalkanes. The antioxidant activity of rosemary extract has generally beenattributed to the presence of three phenolic compounds: carnosic acid(CA), carnosol (CAR) and rosmarinic acid (Cuvelier et al., 1994; Frankelet al., 1996).

Rosemary plants, like many other Lamiaceae family species, grown underhigher temperatures, light intensity and longer day length periods tendto have increased levels of monoterpenes. The harvest time is crucial tomaximize the biomass and carnosic acid concentration in rosemary. Todate, progressive genetic improvement has not yet been made in theimprovement of rosemary for better production of carnosic acid or otherantioxidant molecules. It has been reported that wild type ornaturally-occurring rosemary accumulates approximately 2-3% carnosicacid on a dry matter basis. Extraction of carnosic acid from ahyper-accumulating rosemary clonal line is crucial for producingcommercially viable rosemary extract rich in carnosic acid.

The present invention, KI937, is a proprietary rosemary clonal line withgood agronomic characteristics that remarkably produces more than 9%carnosic acid, and more particularly 9.8% on a dry matter basis underfield conditions. K1937 is a first-generation carnosic acidhyper-accumulating rosemary clonal plant line with substantialcommercial potential.

SUMMARY OF THE INVENTION

The present invention relates to a plant or clonal line of Rosmarinusofficinalis L. named KI937 that has elevated carnosic acid levels,excellent vigor and overall robust agronomic traits. The presentinvention is an upright perennial clonal line that was developed from arosemary breeding program at Kemin Industries.

The present invention, with elevated levels of carnosic acid and goodgrowth habits, was selected through a robust breeding program and hasbeen asexually propagated to produce a clonal line of identical plants.

Plants of the cultivar KI937 have not been observed under all possibleenvironmental conditions. The phenotype may vary somewhat withvariations in environment and culture such as temperature, lightintensity, day length, water status, and/or fertilizer rate or typewithout, however, any variance in genotype.

An object of the present invention is a plant with a high level ofcarnosic acid for use as an antioxidant in human and animal food,beverages and personal care products.

Another object of the invention is a variety of rosemary that is novel,stable, and uniform and has good agronomic characteristics that permitefficient cultivation of the variety as a crop that produces a highamount of biomass from which carnosic acid can be extracted.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 depicts the frequency distribution for carnosic acid accumulationin breeding population.

FIG. 2 depicts carnosic acid content in select lines from South Texas.

FIG. 3 depicts carnosic acid content in select lines in New Mexico.

FIG. 4 shows the breeding scheme for development of KI937.

FIG. 5 summarizes the scale-up process of KI937.

FIG. 6 shows the length of a leaf of a plant of the variety KI937against a centimeter scale.

FIG. 7 shows the width of a leaf of a plant of the variety KI937 againsta centimeter scale.

FIG. 8 shows a lateral branch of the variety KI937.

FIG. 9 depicts the phylogenetic tree based on 3068 SNPs generated bytGBS shows clustering of eight rosemary clonal lines along with publicvariety Arp.

FIG. 10 depicts the phylogenetic tree based on 120 KASP marker genotypedata shows clustering of Kemin proprietary rosemary clonal lines andpublic variety Arp.

FIG. 11 represents the factorial analysis from the dissimilarity indexfor rosemary clonal lines using 120 KASP markers.

DETAILED DESCRIPTION OF THE INVENTION

Rosemary clonal line KI937 is a proprietary clonal line developedthrough selective crossbreeding.

Methods and Materials: A crossing block containing rosemary plantsobtained from Papa Geno's herb farm in Nebraska United States and plantsgenerated from seeds obtained from publicly available sources wasestablished at Kemin Des Moines based greenhouse. Mature floweringplants were assembled in a bee cage in October 2009. Bees wereintroduced to aid with pollination. Seed that represented either a selfor a cross were collected from each flowering plant in the bee cageduring February 2010. In addition, since dried rosemary flowers hadfallen on the floor of the bee cage, seed from the floor that wouldstill represent a self or a cross from plants within the bee case wascollected.

More than 2000 seeds from the crossing block were planted in agreenhouse located on the campus of Kemin Industries, Inc. in DesMoines, Iowa. From these seeds, 380 seedlings grew well and were grownto full maturity under greenhouse conditions. When plants were 8 to 10inches tall with a minimum of 4-5 branches, biomass from leaves andyoung stems representing new growth were sampled from each plant duringDecember 2010. Leaf samples from each plant were collected at the timeof maturity and air dried for CA analysis. Dry leaf samples from eachline were quantitated for CA content using a previously developed methodfor quantitating carnosic acid on a dry mater basis.

A shorter HPLC assay limited to CA and CAR determination with a shorterHPLC assay time of 17 minutes suitable for screening large numbers ofsamples was used to analyze all the samples. Samples were prepared fromeach rosemary tissue and three successive extractions from the samesample were carried out. CA and CAR content were determined based on dryweight in all the samples in triplicate from both experiments. Extensivevariation for carnosic acid accumulation was seen among the individualswithin the population (FIG. 1).

Only twenty-nine lines with CA levels greater than 6% on dry matterbasis were identified, of which only thirteen lines had CA levels higherthan 7%. Most of the population was on the lower end of carnosic acidaccumulation spectrum.

Seventy-seven lines designated as high-CA lines were selected forfurther field evaluation. These plants were grown in the greenhouse toallow for new growth. Cuttings were taken from each individual anddipped in rooting hormone (Dip'N Grow); and stuck in propagation traysfilled with soilless mix. Propagules rooted after 8 to 10 weeks to beconsidered a quality plug for field transplanting. Each clonallypropagated line was transplanted in 20 ft single strip rows in a fieldlocation in a south Texas, for initial field evaluation during October2013. Adequate care including irrigation, fertilizer and weed controlwas given to help the propagates establish in plots. Beginning April2014 to August 2015, leaf and stem tissue was sampled from the top 6inches of new growth of rosemary lines established in field plots. Leaftissue was air-dried and tested in the lab for CA content. K10937 alongwith a few other lines were identified as high-CA accumulation lineswith CA content>8% on a dry mater basis (FIG. 2). KI937 in particular,showed an average of 8.6±0.8% CA content throughout the sampling periodin 2014 through 2015.

Two years after establishing the plots in South Texas excessive rainsdestroyed the plots. These lines were again propagated during the wintermonths of 2014-2015 in the Kemin greenhouse. Plugs were transplantedinto 20-foot single row plots in the eastern part of New Mexico duringApril 2015. Adequate care including irrigation, fertilizer and weedcontrol was given to help the propagates establish in plots.

From August 2016 through September 2018, leaf and stem tissue weresampled from the top 6 inches of new growth of the rosemary clonallines. Biomass from selected lines were harvested at appropriate cropmaturity and air-dried for CA quantitation. KI937 continued to show thehighest level of carnosic acid accumulation on a dry matter basis (FIG.3). The average CA level in KI937 eras 10.05%±0.9% under fieldconditions, which is extremely unusual under field conditions. To date,KI937 has surpassed all tested rosemary clonal lines, for carnosic acidaccumulation. Carnosic acid levels were generally consistent acrosslocations. In this study, despite the significant location effect andgenotype x location effect, KI937 was found to be have higher CA contentin both locations (FIGS. 2 and 3).

The present invention, the plant or clonal line designated as KI937 is ahalf-sib rosemary clonal line generated from the cross between KI-R0015and another parent that was in the same bee cage; but it is not clearfrom which parent would have contributed the pollen (FIG. 4).

KI937 was evaluated in plots for overall vigor, leaf biomass, CA yieldsand winter hardiness along with other lines as shown in Table 1 and 2.KI937 has a consistent average winter hardiness compared to other linesin the plot.

The propagation tests were conducted at the commercial propagator in NewMexico for an initial rooting assessment compared to other lines (Table1). All lines with acceptable rooting levels in combination withelevated CA content and dry leaf biomass yields were advanced toreplicated six row plots (Table 1 and Table 2).

KI937 is a very high carnosic acid accumulating line (hyper-accumulatingline) and is one of only a few to accumulate CA to this level to date.KI937 offers an economically viable source of carnosic acid extractionand is a significant improvement over other known or commercially grownrosemary lines used for antioxidant extraction purposes.

TABLE 1 Selected advanced rosemary clonal lines for replicated plotevaluation Winter Average Hardiness Rooting Test CA % Plant ID 2016-20182016 2016-2018 KI937 Medium 62%  10 ± 0.5 KI-R1597 Medium 90% 8.2 ± 0.3KI-R1054 Less 75% 7.1 ± 0.5 KI1005 Medium 59% 7.1 ± 0.8 KI-R0936 Medium86% 6.9 ± 0.7 KI-R1566 Excellent 90% 6.4 ± 0.5 KI-R0998 Good 50% 6.3 ±0.4 KI-R0633 Good 30% 6.2 ± 0.7 KI-R1621 Medium 91% 6.2 ± 0.7 KI-R1549Excellent 90% 5.5 ± 0.9 KI28 Medium 48% 4.7 ± 0.7

TABLE 2 Biomass and CA yield of selected advanced rosemary clonal linesEstimated Dry Leaf (Kg/Acre) Estimated CA Yield (Kg/Acre) Plant ID 20162017 2018 2016 2017 2018 KI937 395.9  191.6 ± 76.3   714.3 ± 138.7 39.619.1 ± 7.6  73.2 ± 14.5 KI-R1597 709.3 — 1285.6 ± 219.9 55.2 — 108.6 ±18.2 KI-R1054 520.6  212.7 ± 5.5    985.3 ± 121.2 38.1 13.7 ± 0.4  72.2± 8.8  KI1005 518  271.4 ± 43    1175.6 ± 255.8 39.2 16.3 ± 2.6  92.1 ±20   KI-R0936 374.5  257.7 ± 36.2   962.7 ± 190.8 26.5 18.3 ± 2.5  71.7± 14.4 KI-R1566 —  578.6 ± 95.3  1956.5 ± 263.6 — 35.4 ± 5.7 133.7 ±18.1 KI-R0998 511.2  355.3 ± 49.8  1287.7 ± 164.8 30.7   22 ± 3    88.6± 11.2 KI-R0633 528.3  359.9 ± 74.2  1382.2 ± 215.3 32.6 19.3 ± 4   94.2 ± 14.6 KI-R1621 598.6  211.9 ± 76    1089.6 ± 231   38.5 11.9 ±4.3  71.8 ± 15.3 KI-R1549 — 1172.3 ± 298.9 2964.2 ± 181.5 —  62.5 ± 15.1181.5 ± 11.1 KI28 392.5  236.1 ± 58.2   949.3 ± 89.1  16.3 11.4 ± 2.9 51.8 ± 4.8 

Based on the above data, KI937 was identified for large-scale plantingbeginning 2019 (FIG. 5). Rosemary cuttings were taken from the fieldduring the growing season and then moved into a commercial propagationgreenhouse. Additional care is required for propagules taken from thefield which are typically slower rooting and heavier disease loadcompared to greenhouse sourced as plants. The successful propagation ofKI937 has improved with time as experienced growers have modified thepropagation protocol to achieve>80% rooting to finished plug for bothsources.

Phenotypic descriptions of traits required for observation are providedin Table 3. Phenotypic characteristics of KI937 are provided in Table 4and FIGS. 6-8 provide visual confirmation for phenotypic characteristicsof KI937.

TABLE 3 Description of the traits for KI937 collected in greenhouse.Trait Description Days to first flowering Number of days fromtransplanting to when the first spike/inflorescence emerges Plant height(cm) Length of the middle stem at the time of flowering Length oflateral branches (cm) Length/Ht of the lateral stems Number of lateralbranches Count on the number of lateral branches Number of nodes on themiddle Count on the number of nodes of the middle stem stem Internodelength on the middle Measure the length between 9^(th) and 10^(th)internode of the middle stem stem Number leaves on the middle Count thenumber of spikes per plant stem Leaf width taken from the fully openedleaf from 10^(th) node of the middle stem Leaf length taken from thefully opened leaf from 10^(th) node of the middle stem Leaf area (cm²)taken from the fully opened leaf from 10^(th) node of the middle stemLeaf to stem ratio Leaves separated from stem and weighed to obtainratio Top 6″ of the plant cut and air dried for 48 hours for carvacrolCarnosic acid content quantitation Ratio of aerial plant parts to Onfresh weight basis roots Estimated dried biomass per Actual driedbiomass obtained from 1 m2 area & estimated on a per acre acre basis

TABLE 4 Phenotypic characteristics of KI937 observed in the greenhouse.Trait Average Range Days to first flowering from date of N/A N/Atransplanting rooted Plant height (cm) 40 cm 30-50 cm Length of lateralbranches (cm) 22.5 cm 15-30 cm Number of lateral branches 32.5 25-40Number of nodes on the middle stem 16 14-18 Internode length on themiddle stem 2.4 cm 1.2-1.7 cm Number of leaves on the middle stem 348216-480 Leaf width 0.25 cm 0.2-0.3 cm Leaf length 2.55 cm 2.0-3.1 cmLeaf area (cm²) 0.67 cm² 0.4-0.93 cm² Leaf to stem ratio 3.7 3.5-3.9Carnosic acid content (%) 10.5%   10.2-10.8% during maturity Drybiomass/acre 357.1 Kg 196.0-518.3 Kg

DNA Fingerprint Profile Through Single Nucleotide Polymorphism (SMPs)And KASP Markers:

In addition to phenotypic observations, the rosemary clonal plant lineKI937 can also be identified by its genotype. DNA based techniques aremore powerful in identifying the genetic differences between clonallines varieties. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same variety ora related variety or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques varioustechniques. SNPs are one of the most abundant DNA variants related togenotype, chemotype and phenotype found in plant genomes. SNP basedmarker assays are high throughput, highly reproducible and highlyapplicable in generating DNA fingerprints for a plant species ofinterest. Development of SNP markers for fingerprinting requires thefollowing steps: (i) partial sequencing of multiple clonal lines bynext-generation sequencing technologies; (ii) SNP discovery afteraligning sequence reads; (iii) validate SNPs to identify true variants;and (iv) SNP genotyping of germplasm collection or populations to createa unique fingerprint based on polymorphisms. A subset of Keminproprietary rosemary lines and publicly available rosemary clonal lineswere sequenced to identify SNPs and validate them. These SNPs wereconverted to the KASP marker system in order to genotype Kemin'sproprietary germplasm to identify true allelic variants and create aunique genetic fingerprint for each of the rosemary clonal line underinvestigation.

Sequencing and SNP identification. Sequencing, read quality checking,read trimming, de novo read alignment, and identification of SNPs werecarried out by a third-party service provider Dat2Bio LLC in Ames, Iowa.Leaf samples of eight proprietary rosemary clonal lines and publiclyavailable rosemary line such as ‘Arp’ were obtained from the Kemingreenhouse, frozen/freeze-dried and provided to Dat2Bio. The eightclonal lines were replicated 12 times to make a total 96 sequencingsamples for maximum coverage of whole genome sequence. Sequencing wasperformed using LifeTech's Ion Proton instrument. Sequence reads werefiltered using standard filtering parameters to remove the poor-qualityreads and sorted with barcodes that represent individual sample. Trimmedclean reads were then used for de novo genome assembly of rosemarygenome. Tunable Genotyping by Sequencing (tGBS) was used to obtainsequence information and discovery of SNPs. Two different approacheswere used for SNP calling, (i) Stringent Minimum Call Rates 50% (MCR50),in which all the SNPs were identified with a ≥50% minimum call rate withan average 85 reads per SNP position, and (ii) Stringent Minimum CallRates ≥75% (MCR75) in which ≥75% minimum call rate with an average 85reads per SNP position were used.

The raw tGBS reads from the Ion Proton instrument were trimmed andaligned to create a de novo assembly of rosemary genome, since no priorgenome information exists for rosemary. Aligned sequences were used todiscover single base differences among eight clonal lines to identifySNPs, which were filtered resulting in 181,618 SNPs. From these, therewere at least ˜11,300 polymorphic SNPs between KI937 and Arp. Fromthese, a set of 144 SNPs that can distinguish all the eight clonal lineswas identified and KASP markers were developed for the same.

DNA extraction. All rosemary clonal lines were genotyped using 144 KASPmarkers. Leaf tissue collected from all the clonal lines was freezedried and ground into fine powder in 1.5 ml Eppendorf tubes, and DNA wasisolated using CTAB protocol with the following modifications. Freshlyprepared 2X CTAB with 2% PVP, 0.2% Mercaptoethanol and 5 μl RNaseA wasadded to the ground tissue samples and immediately placed in a waterbath for incubation at 65° C. for 90 mins with occasional shaking of thetubes. Equal volume of chloroform isoamyalcohol was added and shaken for10 mins for protein precipitation followed by centrifugation at 12000rpm for 10 mins. Supernatant was transferred into new 1.5 ml tubes andDNA was precipitated with 0.6 volume 2-propanol followed by washing DNApellet with 75% ethanol. Air dried pellet was dissolved in RO water forKASP assays.

Development of KASP markers genotyping. A total of 3068 SNP markers wereidentified through the tGBS pipeline among 8 rosemary clonal linesincluding public variety ‘Arp’. Of these 3068 SNPs, based on contiglength and reliability of SNP calls, 144 KASP markers were designed andoligo-synthesized. All of the 144 KASP markers were used to assay 18clonal lines including 8 clonal lines that were used for original tGBSgenotyping.

Genotyping and data analysis. For each sample genotyping was performedin 5 μl KASP PCR reactions using Roche Light Cycler® 480 qPCR machinewith 384 well plates (Roche Life technology USA). KASP PCR reactionscontained 2.5 μl 4X KASP master mix (LGC Genomics), 0.08 μl primer mix(IDT, IA, USA) and 2.5 μl template DNA. Thermocycling was started with15 min at 94° C., a touchdown phase of 10 cycles at 94° C. for 20 s and65° C. for 60 s with a 1° C. decrease in temperature per cycle, followed35 cycles of 94° C. for 20 s and 55° C. for 60 s. Once the thermal cyclewas complete, fluorescence signal was acquired at 520 nm (FAM) and 556nm (HEX) at 37° C. for 10 s. Data was then analyzed using LightCycler®480 SW 1.5.1 software (Roche Life Science, Calif., USA) to identify SNPgenotypes. For each marker, homozygous and heterozygous calls were madeand scored as co-dominant markers.

KASP genotyping assay results were recorded as a two-letter codes i.e.,AA, BB, AB or No calls. A DNA fingerprint was made using all the SNPbased KASP marker genotypes. AA represents homozygous reference allele,BB represents homozygous alternate allele, AB represents heterozygousand No calls represents absence of both alleles for respective KASPmarker. Phylogenetic tree and factorial analysis from the dissimilarityindex were carried out using software, DarWin. Cluster analysis andfactorial analysis of dissimilarity were calculated usingNeighbor-Joining method. Multiple rooted phylogenetic trees weregenerated using DarWin software. Heatmap analysis and genetic diversityanalysis were performed using software R with statistical packagesheatmap and SNP ready, respectively.

A total 246,336 scaffolds were generated ranging from 30 by to 203 bplong. An average 12,112,651 reads were achieved for each clonal line.Genome assembly of short reads generated 21,830,060 bp which was usedfor SNP calling using tGBS pipeline. SNP calling was performed andcompared using two difference approaches, (i) Stringent Minimum CallRates 50% (MCR50), in which all the SNPs were identified with a ≥50%minimum call rate with an average 85 reads per SNP position and (ii)Stringent Minimum Call Rates ≥75% (MCR75) was used in which a total 3068SNPs were identified.

The eight clonal lines were clustered into two major clades based on the3068 SNPs generated by tGBS system. Arp and the three commercial lines(KI28, KI937 and KI1005) were placed in a distinctly different clade(FIG. 9).

Cluster Analysis and phylogenetic tree construction using KASP markerdata. Eighteen clonal lines were clustered into groups based on theirsimilarities in KASP genotype data. Genotype data from a total 120 KASPmarkers were used for generating clades/clusters. Cluster analysis ofKASP marker variation corresponded well to the SNP variation with moreresolution on clusters (FIG. 10). RASP marker data clustering resultedin three distinct clades (FIG. 10). Arp was distinctly different fromall the lines arid formed its own clade. The second clade consisted ofvarious sub-clusters, of which KI937 and KI1005 formed asub-sub-cluster. Greater level of similarity between KI28 and KI633clustered them together, and relatively closer to KI1005 and KI937. Thefirst clade generally consisted of commonly available rosemary linessuch as KI-R0001 (public name Nancy Howard), KI-R007 (public name PineScented), KI-R0042 (public name Hulka) and KI-R0073 (public name Baby PJrosemary) along with a few Kemin breeding lines, and further dividedinto three sub clusters. KI-R0001 formed a distinct sub-clade within thefirst clade. Three other public varieties (KI-R0007, KI-R0042,KI-R0073), along with a breeding line KI1346 formed a sub-cluster withinthe first clade.

Similarity coefficients of genetic distance between clonal lines rangedfrom −0.02 to 0.35 (FIG. 11). The greatest degree of dissimilarity wasobserved between Arp and KI937 (FIG. 11).

A SNP and KASP marker profile/genetic fingerprint were created for eachof the Kemin rosemary germplasm. Due to the inherent geneticdifferences, Kemin's proprietary rosemary lines were distinctly groupedinto specific clades. More importantly, a distinct DNA-based markerprofile for KI937 in comparison with the publicly available andcommercially grown clonal line Arp has been created. A set of KASPmarkers were identified that can differentiate KI937 from Arp is shownin Table 5.

TABLE 5 KASP marker genotype data distinguish Arp from KI937 and otherlines KASP Markers KI28 KI937 KI1005 Arp P24 AA AA AA AB P34 BB AA AA ABP47 BB BB AA AB P53 AB BB BB AA P58 AB AA AA BB P62 BB BB BB NA P63 ABAB AB AA P66 AA AB AB AA P71 NA BB BB NA P72 AA AB AB AA P75 AA BB AA ABP76 AB AB AB BB P77 AB AB AB BB P78 AA AB AB AA P79 BB AB AB BB P80 BBBB BB AB P83 AB AB AA BB P84 AB AB AA BB P92 AA AA AA AA P103 AA AB ABAA P108 AA AB AB AA P115 AA AB AB AA P116 AA AB AB AA P118 AA NA AA ABP123 AB BB BB AA P129 AA AA AA AB P131 AA AA AA AB P138 BB AB AB BB

The occurrence of high carnosic acid accumulating lines with the DNAmarker-profile, such as described herein, are exceedingly rare. The fewknown rosemary plants capable of producing carnosic acid levels in leaftissue (e.g., greater than 5% carnosic acid on a dry weight basis) diewithin a few months. In contrast, the present invention, plant or clonalline denominated KI937, has survived for four years in the field and itsvigor and biomass is equivalent to other commercial varieties.

According to at least one embodiment of the present invention, therosemary plant leaf tissue contains at least 9% carnosic acid, forinstance at least 9.8% carnosic acid or at least 10% carnosic acid, forinstance at least 10.05% on a dry weight basis.

In another embodiment, the rosemary plant leaf tissue contains up to 20times the amount of carnosic acid compared to known rosemary plants thatmay range from 0.5% to 7% carnosic acid on a dry weight basis.

The present invention is a unique, genetically distinct and stableclonal line, capable of accumulating more than 9%, and in some instancesproduced more than 9.8% or 10% carnosic acid during the active growingseason, under field conditions. In contrast to the present invention,the average level of carnosic acid observed in an unselected populationof rosemary is about 3% on a dry matter basis; the average level ofcarnosic acid observed in another commercial line, K128, is about 5.5%on average on a dry matter basis under field conditions. Combined withgood growth habits and biomass, KI937 is a unique and highly desirablebiomass source for carnosic acid extraction.

Evidence of Uniformity and Stability

No variants of any kind have been observed since the variety KI937 wasidentified, indicating the stability and uniformity of the genotype. Itis clear from these results that the KI937 cultivar is stable andreproduces true to type in successive generations of asexualreproduction.

Statement of Distinction

KI937 consistently produces higher per dry weight levels of carnosicacid as compared to other selected and commercial varieties, forinstance up to 20 times the amount of carnosic acid on a dry weightbasis, while its vigor and biomass is equivalent to other commercialvarieties. Due to vigorous vegetative growth this genotype can beharvested multiple times in a season and has the potential of growing inany temperate climate.

Deposit Information

Applicant made a deposit of rosemary plant line KI937 with theProvasoli-Guillard National Center for Marine Algae and Microbiota,Bigelow Laboratory for Ocean Science, 60 Bigelow Drive, East Boothbay,Me. 04544, assigned Accession Number 201909053, from which plants ofrosemary plant line KI937 can be reproduced. The biological tissue wasdeposited on Sep. 24, 2019 and was obtained from the line maintained byKemin Industries, Inc., 1900 Scott Avenue, Des Moines, Iowa 50317, sinceprior to the filing date of this application. Access will be availableduring the pendency of the application to the Commissioner of Patentsand Trademarks and persons determined by the Commissioner to be entitledthereto upon request. Upon allowance of any claims in the application,the Applicant will make the deposit available to the public pursuant to37 C.F.R. § 1.808. This deposit of rosemary plant line KI937 will bemaintained the National Center for Marine Algae and Microbiota, which isa public depository, for a period of 30 years, or 5 years after the mostrecent request, or for the enforceable life of the patent, whichever islonger, and will be replaced if it becomes nonviable during that period.Additionally, Applicant has or will satisfy all of the requirements of37 C.F.R. §§ 1.801-1.809, including providing an indication of theviability of the sample upon deposit. Applicant has no authority towaive any restrictions imposed by law on the transfer of biologicalmaterial or its transportation in commerce. Applicant does not waive anyinfringement of rights granted under this patent or under the PlantVariety Protection Act (7 U.S.C. § 2321 et seq.).

Having described the invention with reference to particularcompositions, theories of effectiveness, and the like, it will beapparent to those of skill in the art that it is not intended that theinvention he limited by such illustrative embodiments or mechanisms, andthat modifications can be made without departing from the scope orspirit of the invention, as defined by the appended claims. It isintended that all such obvious modifications and variations be includedwithin the scope of the present invention as defined in the appendedclaims. The claims are meant to cover the claimed components and stepsin any sequence which is effective to meet the objectives thereintended, unless the context specifically indicates to the contrary.

The foregoing description has been presented for the purposes ofillustration and description. It is not intended to he an exhaustivelist or limit the invention to the precise forms disclosed. It iscontemplated that other alternative processes and methods obvious tothose skilled in the art are considered included in the invention. Thedescription is merely examples of embodiments. It is understood that anyother modifications, substitutions, and/or additions may be made, whichare within the intended spirit and scope of the disclosure. From theforegoing, it can be seen that the exemplary aspects of the disclosureaccomplishes at least all of the intended objectives.

1. A method of producing carnosic acid, comprising extracting carnosicacid from plant tissue of a rosemary plant denominated KI937 as producedby a rosemary line deposited with the National Center for Marine Algaeand Microbiota and assigned Accession Number
 201909053. 2. The method ofclaim 1, wherein the plant tissue is selected from the group consistingof leaf, rhizome, root, seed, or stem tissue.
 3. The method of claim 1,further comprising using the extracted carnosic acid as an antioxidantin a product selected from the group consisting of human food, animalfood, beverages and personal care products.
 4. A method of providing anantioxidant, comprising extracting carnosic acid from plant tissue of arosemary plant denominated KI937 as produced by a rosemary linedeposited with the National Center for Marine Algae and Microbiota andassigned Accession Number
 201909053. 5. The method of claim 4, whereinthe antioxidant is included in product selected from the groupconsisting of: human food, animal food, beverages and personal careproducts.
 6. A method of providing an antioxidant, comprising: a.growing a rosemary plant denominated KI937 as produced by a rosemaryline deposited with the National Center for Marine Algae and Microbiotaand assigned Accession Number 201909053; b. harvesting the plant tissueof the rosemary plant; c. extracting carnosic acid from the planttissue; and d. using the carnosic acid in a product.
 7. The method ofclaim 6, wherein the tissue is selected from the group consisting ofleaf, rhizome, root, seed, or stem tissue.
 8. The method of claim 6,wherein the antioxidant product is selected from the group consisting ofhuman food, animal food, beverages and personal care products.
 9. Themethod of claim 6, wherein the plant tissue comprises at least 9%carnosic acid on a dry weight basis after the plant material is dried.10. A method by which the rosemary plant line denominated KI937 is usedin a breeding program by using pollen from said clonal lines as a maleparent; or flowers of said clonal lines as a female parent, for thegeneration of seed of a breeding population from which furtherselections can be made.
 11. A plant or node of the rosemary plant linedenominated KI937, or a cutting or part thereof, wherein representativeplant tissue of KI937 has been deposited with the National Center forMarine Algae and Microbiota and assigned Accession Number
 201909053. 12.The plant of claim 11, wherein the plant or part thereof includes aleaf, rhizome, root, seed, or stem tissue.
 13. The plant or node ofclaim 11, wherein the plant, node, cutting or part of the plant are usedas a starting material to grow additional plants.
 14. Pollen of theplant of claim
 11. 15. An ovule of the plant of claim
 11. 16. A methodfor producing rosemary seed, said method comprising the steps of: (i)growing one or more plants from one or more of the plant or node ofclaim 11; (ii) pollinating one or more plants produced from one or moreof the node or plant of claim 11 by self-pollination or by pollinationwith pollen from a different plant and, (iii) harvesting resultant seed,and growing new population.
 17. A rosemary plant or part thereof, havingthe physiological, morphological characteristics and DNA profile of therosemary plant line denominated KI937 that has been deposited with theNational Center for Marine Algae and Microbiota and assigned AccessionNumber
 201909053. 18. The rosemary plant of claim 17, wherein the plantor part thereof includes plant tissue, node, leaf, rhizome, root, seed,or stem tissue.
 19. Pollen of the rosemary plant of claim
 17. 20. Anovule of the rosemary plant of claim 17.